This project will use a multidisciplinary team of molecular, structural, and computational biologists together with medicinal chemists to develop and evaluate compounds that have inhibitory activity against the flavivirus genus of plus strand RNA viruses. These compounds will be used to probe the function of their target proteins in the life cycle of the virus. This project builds on existing collaborations and extends previous observations by the Purdue group. The flaviviruses contain members of MAID Priority Pathogens that are found in all three categories A-C. The focus will be primarily on dengue virus, a category A pathogen, because of both its medical importance and the substantial structural data and reagents that we and others have compiled over the last several years. We will combine both foundational basic research, together with a translational component that involves in vitro and in vivo compound evaluation to identify small molecules suitable for pre-clinical antiviral development. The approaches used to identify such compounds will also yield insight into the assembly and replication of flaviviruses. We will determine the conformational states of the flavivirus structural proteins as they progress in the immature virion to the mature state, and evaluate the ability of compounds to interfere with these transitions and the process of viral entry. We will evaluate the dynamic states of the flavivirus particle, the structural transitions that occur during maturation and entry, and the binding of small molecule inhibitors to the envelope proteins. Two sets of inhibitory molecules have been developed based on predicted binding to 1) an E protein n-octyl-b-Dglucoside (BOG) binding pocket;and 2) sites on E that are involved in protein - protein interactions. These two sets serve as starting reagents in compound evaluation and further development. We will also identify compounds that inhibit flavivirus genome replication, identify their mechanism of inhibition, and evaluate their efficacy in an animal model. A high throughput flavivirus replicon screen was used to identify small molecule inhibitors of replication. Targets of these inhibitors will be identified by genetic selection, initial hits will be expanded by successive rounds of medicinal chemistry, and the compounds will be evaluated both in vitro using cell culture models and in vivo using small animal models.